Miniaturised immobilised enzymatic reactors can be used for small scale digestion of proteins. There is need for such devices; small scale devices are used either for processing of analytical sample quantities, or as proof of concept before protein digestion at larger scale. This application note compares the performance of a flow through miniaturised immobilised enzymatic reactor (μIMER) with in-solution batch digestion of simple proteins and complex matrices. Automation of peptide analysis by coupled LC-MS is explored as an option to increase throughput. In the cases evaluated, the miniaturised immobilised enzymatic reactor offered comparative results to overnight in-solution digestion, within less than 10 minutes.
Pre-activated CIMmic™ monolithic columns with 100 μL bed volume were immobilised with trypsin from bovine pancreas. This small format allows coupling to HPLC for on-line protein digestion, as well as syringe (manual) operation of the IMER. Pre-treated samples (denatured, alkylated and ultra-filtered) are injected into the column, and the eluate (tryptic digests) are subjected to LC-ESI-MS-MS analysis for protein identification and post-translational modification (PTM) determination.
Pre-activated CIMmic™ Monolithic Columns are used as a basis for preparation of small volume affinity chromatographic columns as well as enzyme reactors. Small bed volume and flexible design makes them a powerful tool for screening purposes and immobilization protocol optimizations. Range of covalently bound ligands is wide and includes diverse set of proteins, peptides, nucleotides and other affinity ligands. The covalent nature of the bond between the ligand and matrix reduces leaching and improves stability and reusability. Reaction conditions must cater to their specific physiochemical nature.
Successful preparation of an affinity column includes a decision on the appropriate matrix chemistry and determination of an optimal immobilization protocol. Presented case study explores the basics of a coupling protocol optimization using covalent immobilization of Recombinant Prokaryotic Lectins (RPL-Gal1) on CIMmic CDI-0.1 and CIMmic ALD-0.1 columns, as an example. Carboxy imidazole (CDI) and aldehyde (ALD) activated CIMmic™ columns are used for covalent immobilization of amine or thiol containing molecules.
The application describes separation of Ni species by assembling four weak CIM DEAE anion-exchange disks into a monolithic column. The concentrations of the Ni species eluted from the column were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID)-ICP-MS. The Ni binding ligands eluted under the chromatographic peaks were identified off-line by tandem electro spray mass spectrometry (ESI-MS-MS), scanning for negative ions.
The mild chromatographic conditions of the CIM DEAE disks preserved chemical species and enabled separation of negatively charged Ni complexes.4 NH4NO3 was chosen as eluent since it enabled separation of Ni species and is compatible with ICP-MS and mass spectrometry detectors.
PEGylation involves the formation of a stable covalent bond between activated poly (ethylene glycol) polymers and polypeptidic drugs and molecules. This process causes a change in protein hydrophobicity and results in variance between the obtained conjugates. Despite this, hydrophobic interaction chromatography (HIC) is used less frequently for separation of PEGylation reaction products than other techniques. Separation of PEGylated conjugates of Ribonuclease A (RNase A) via HIC on monolithic supports was analysed in this work. The protein was PEGylated in the N-terminal amino group with 20 kDa methoxy poly (ethylene glycol) propionaldehyde.
A supernatant from Phanerochaete chrysosporium cultivation was loaded on CIM® QA Disk, and elution was effected by a linear gradient at a flow rate of 3 mL/min (9 CV/min). Baseline separation of isoenzymes H2, H6/H7, H8 and H10 was achieved in less than 3 minutes.